Lysosomal Storage Disease
The lysosomal compartment functions as a catabolic machinery that degrades waste material in cells. Degradation is achieved by a number of hydrolases and transporters compartmentalized specifically to the lysosome. There are today over 40 identified inherited diseases where a link has been established between disease and mutations in genes coding for lysosomal proteins. These diseases are defined as lysosomal storage diseases (LSDs) and are characterized by a buildup of a metabolite (or metabolites) that cannot be degraded due to the insufficient degrading capacity. As a consequence of the excess lysosomal storage of the metabolite, lysosomes increase in size. How the accumulated storage material cause pathology is not fully understood but may involve mechanisms such as inhibition of autophagy and induction of cell apoptosis (Cox & Cachón-González, J Pathol 226: 241-254 (2012)).
Enzyme Replacement Therapy
Storage can be reduced by administration of a lysosomal enzyme from a heterologous source. It is well established that intravenous administration of a lysosomal enzyme results in its rapid uptake by cells via a mechanism called receptor mediated endocytosis. This endocytosis is mediated by receptors on the cell surface, and in particular the two mannose-6 phosphate receptors (M6PR) have been shown to be pivotal for uptake of certain lysosomal enzymes (Neufeld; Birth Defects Orig Artic Ser 16: 77-84 (1980)). M6PR recognize phosphorylated oligomannose glycans which are characteristic for lysosomal proteins.
Based on the principle of receptor mediated endocytosis, enzyme replacement therapies (ERT) are today available for six LSDs, (Gaucher, Fabrys, Pompe and the Mucopolysaccharidosis type I, II and VI). These therapies are efficacious in reducing lysosomal storage in various peripheral organs and thereby ameliorate some symptoms related to the pathology.
A majority of the LSDs however cause lysosomal storage in the central nervous system (CNS) and consequently present a repertoire of CNS related signs and symptoms. A major drawback with intravenously administered ERT is the poor distribution to the CNS. The CNS is protected from exposure to blood borne compounds by the blood brain barrier (BBB), formed by the CNS endothelium. The endothelial cells of the BBB exhibit tight junctions which prevent paracellular passage, show limited passive endocytosis and in addition lack some of the receptor mediated transcytotic capacity seen in other tissues. Notably, in mice M6PR mediated transport across the BBB is only observed up to two weeks after birth (Urayama et al, Mol Ther 16: 1261-1266 (2008)).
Glycosylation of Lysomal Enzymes
In general, N-glycosylations can occur at a Asn-X-Ser/Thr sequence motif. To this motif the initial core structure of the N-glycan is transferred by the glycosyltransferase oligosaccharyltransferase, within the reticular lumen. This common basis for all N-linked glycans is made up of 14 residues; 3 glucose, 9 mannose, and 2 N-acetylglucosamine. This ancestor is then converted into three general types of N-glycans; oligomannose, complex and hybrid (FIG. 7), by the actions of a multitude of enzymes that both trim down the initial core and add new sugar moieties. Each mature N-glycan contains the common core Man(Man)2-GlcNAc-GlcNAc-Asn, where Asn is the attachment point to the protein.
In addition, proteins directed to the lysosome carry one or more N-glycans which are phosphorylated. The phosphorylation occurs in the Golgi and is initiated by the addition of N-acetylglucosamine-1-phosphate to C-6 of mannose residues of oligomannose type N-glycans. The N-acetylglucosamine is cleaved off to generate Mannose-6-phosphate (M6P) residues, that are recognized by M6PRs and will initiate the transport of the lysosomal protein to the lysosome. The resulting N-glycan is then trimmed to the point where the M6P is the terminal group of the N-glycan chain. (Essentials of Glycobiology. 2nd edition. Varki A, Cummings R D, Esko J D, et al, editors. Cold Spring Harbor (N.Y.): Cold Spring Harbor Laboratory Press; 2009.)
The binding site of the M6PR requires a terminal M6P group that is complete, as both the sugar moiety and the phosphate group is involved in the binding to the receptor (Kim et al, Curr Opin Struct Biol 19(5):534-42 (2009)).
Enzyme Replacement Therapy Targeting the Brain by Glycan Modification
A potential strategy to increase distribution of lysomal enzyme to the CNS has been disclosed in WO 2008/109677. In this published application, chemical modification of β-glucuronidase using sodium meta-periodate and sodium borohydride is described (see also Grubb et al, Proc Natl Acad Sci USA 105: 2616-2621 (2008)). This modification, consisting of oxidation with 20 mM sodium periodate for 6.5 h, followed by quenching, dialysis and reduction with 100 mM sodium borohydride overnight (referred to hereinafter as known method), substantially improved CNS distribution of β-glucuronidase and resulted in clearance of neuronal storage in a murine model of the LSD mucopolysaccharidosis VII. Although the underlying mechanism of brain distribution is unclear, it was noted that the chemical modification disrupted glycan structure on β-glucuronidase and it was further demonstrated that receptor mediated endocytosis by M6PR was strongly reduced.
The chemical modification strategy has been investigated for other lysosomal enzymes. For example, modification according to the known method did not improve distribution to the brain of intravenously administrated protease tripeptidyl peptidase I (Meng et al, PLoS One (2012)). Neither has satisfactory results been demonstrated for sulfamidase. Sulfamidase, chemically modified according to the known method, did indeed display an increased half-life in mice but no effect in the brain of MPS IIIA mice. The chemically modified sulfamidase did not distribute to the brain parenchyma when given repeatedly by intravenous administration (Rozaklis et al, Exp Neurol 230: 123-130 (2011)).
Thus, there are still no effective ERT for treatment of LSDs with neurological engagement, such as MPS IIIA. Novel sulfamidase compounds that can be transported across the BBB while remaining enzymatically active would be of great value in the development of systemically administrated compounds for enzyme replacement therapies for the treatment of LSDs with CNS related pathology, such as MPS IIIA.